Composite

Part:BBa_K5165002

Designed by: Ashley Yu, Ziyang Yin, Amy Wang   Group: iGEM24_Concordia-Shanghai   (2024-10-01)
Revision as of 03:57, 2 October 2024 by Davystar (Talk | contribs)


PHB synthesis and depolymerization to BHB

Polyhydroxybutyrate (PHB) can be depolymerized to form the high-value product beta-hydroxybutyrate (BHB). This construct accomplishes BHB formation through two steps in E. coli: 1) PHB synthesis 2) PHB depolymerization The construct is designed so that the host cell prioritizes the production of the enzymes involved in PHB synthesis. Quorum sensing mechanisms, regulated by the lux system, are responsible for transitioning the cell into stage two for PHB depolymerization. Such a two-part system allows for optimized production in each step separately, which is essential for BHB synthesis as the initiation of step two depends on the products of step one.

The first part is regulated by the repressible promoter pTet. PHB synthesis is driven by three enzymes that are characterized in the part BBa_K934001: phaC, phaA, and phaB. The phaCAB enzymes, accompanied by the strong RBS BBa_B0034, constitute the main component of part one. BBa_K2260002 codes for a protein tagging the PHB for secretion, and BBa_K4998027 codes for LuxI, an Acyl-Homoserine Lactone (AHL) synthase enzyme. LuxR, working in tandem with AHL to regulate quorum sensing, is constitutively expressed in the construct. As time progresses, the concentration of both phaCAB enzymes and AHL-luxR complexes increase.The AHL-luxR complexes in turn induce the downstream lux promoter pLux, initiating the start of stage two.

In stage two, the pLux promoter regulates the synthesis of the PHB depolymerase, encoded in the part BBa_K5165000, and the TetR repressor. The TetR repressor inhibits the upstream pTet promoter, ceasing the production of stage one proteins, focusing the cellular processes on depolymerizing the PHB molecules produced from stage one.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 998
    Illegal BglII site found at 1823
    Illegal BglII site found at 5410
    Illegal BamHI site found at 6606
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 304
    Illegal NgoMIV site found at 375
    Illegal NgoMIV site found at 975
    Illegal NgoMIV site found at 1287
    Illegal NgoMIV site found at 1566
    Illegal NgoMIV site found at 2218
    Illegal NgoMIV site found at 2240
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4084
    Illegal BsaI site found at 5220
    Illegal BsaI site found at 6571
    Illegal BsaI.rc site found at 5851
    Illegal BsaI.rc site found at 5907


[edit]
Categories
Parameters
None